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Endothelial Dysfunction in Human Disease
J Mol Cell Cardiol 31, 51–60 (1999) Article No. jmcc.1998.0843, available online at http://www.idealibrary.com on
Endothelial Dysfunction: a Novel Therapeutic Target
Endothelial Dysfunction in Human Disease Helmut Drexler and Burkhard Hornig Medizinische Hochschule Hannover (MHH) Carl-Neubergstr.1, 30625 Hannover, Germany H. D B. H. Endothelial Dysfunction in Human Disease. Journal of Molecular and Cellular Cardiology (1999) 31, 51–60. The vascular endothelium plays a key role in the local regulation of vascular tone by the release of vasodilator substances (i.e. endothelium-derived relaxing factor (EDRF=nitric oxide, NO) and prostacyclin) and vasoconstrictor substances (i.e. thromboxane A2, free radicals, or endothelin). Using either agents like acetylcholine or changes in flow to stimulate the release of EDRF (NO), clinical studies have revealed the importance of EDRF in both basal and stimulated control of vascular tone in large epicardial coronary arteries and in the coronary microcirculation. The regulatory function of the endothelium is altered by cardiovascular risk factors or disorders such as hypercholesterolemia, chronic smoking, hypertension or chronic heart failure. Endothelial dysfunction appears to have detrimental functional consequences as well as adverse longterm effects, including vascular remodelling. Endothelial dysfunction is associated with impaired tissue perfusion particularly during stress and paradoxical vasoconstriction of large conduit vessels including the coronary arteries. These effects may cause or contribute to myocardial ischemia. Several mechanisms may be involved in the development of endothelial dysfunction, such as reduced synthesis and release of EDRF or enhanced inactivation of EDRF after its release from endothelial cells by radicals or oxidized low-density lipoprotein (LDL). Increased plasma levels of oxidized LDL have been noted in chronic smokers and are related to the extent endothelial dysfunction, raising the possibility that chronic smoking potentiates endothelial dysfunction by increasing circulating and tissue levels of oxidized LDL. In heart failure, cytokines and/or reduced flow (reflecting reduced shear stress) may be involved in the development of endothelial dysfunction and can be reversed by physical training. Other mechanisms include an activated renin-angiotensin system (i.e. postmyocardial infarction) with increased breakdown of bradykinin by enhanced angiotensin converting enzyme (ACE) activity. There is evidence that endogenous bradykinin is involved in coronary vasomotor control both in coronary conduit and resistance vessels. ACE inhibitors enhance endothelial function by a bradykinin-dependent mechanism and probably also by blunting the generation of superoxide anion. Endothelial dysfunction appears to be reversible by administering -arginine, the precursor of nitric oxide, lowering cholesterol levels, physical training, antioxidants such as vitamin C, or 1999 Academic Press ACE inhibition. K W: Nitric oxide; Hypercholesterolemia; Heart failure; Flow-dependent dilation.
Introduction The evaluation of endothelial function in humans has focused primarily on endothelial-dependent vasomotion of conduit and resistance vessels, related to the release of nitric oxide (NO) while only recently other important functions of the endothelium have been studied (i.e. adhesive properties of the endothelium with respect to the interaction with leucocytes and platelets (for review see Bevilacqua, 1994) or factors involved in the homeostasis of thrombosis and fibrinolysis (for review see Meidell, 1994). In particular, clinical studies have evaluated vasomotor tone following changes in 0022–2828/99/010051+10 $30.00/0
flow or stimuli for the release of NO from the endothelium, such as acetylcholine, substance P or serotonin. The in vivo assessment of endothelial control of vascular tone in humans is hampered by several limitations and assumptions which need to be considered for the adequate interpretation of clinical studies (for review see Drexler, 1997) Vascular endothelium-dependent tone involves several factors such as nitric oxide, prostaglandins, endothelin or endothelium-derived hyperpolarizing factor. Up to now, the evaluation of endotheliumdependent regulation of vascular tone in patients with cardiovascular disorders focused on the role of NO. Numerous cardiovascular disorders have 1999 Academic Press
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been shown to be associated with altered endothelium-dependent relaxation such as atherosclerosis, diabetes mellitus, smoking, post-menopause state, hypertension, heart failure. Several reviews have discussed these disorders, including the impact of treatment (Drexler, 1997, Celermajer, 1997). This brief review focuses on coronary artery disease and heart failure.
Hypercholesterolemia and coronary artery disease In the clinical setting, hypercholesterolemia is characterized by long-lasting and moderate increases in serum cholesterol and serum cholesterol LDL rather than excessive plasma levels as applied in experimental studies. Nevertheless, clinical studies investigating the effects of hypercholesterolemia on endothelial function in the human circulation have, in general, confirmed the experimental observations that it is is associated with impaired endotheliumdependent vasodilation of coronary and peripheral conduit and resistance vessels (Chowienczyk et al., 1992; Creager et al., 1992; Casino et al., 1993), as tested by endothelium-dependent relaxations in response to acetylcholine or increased flow. In fact, endothelial dysfunction appears to emerge early in humans with hypercholesterolemia, i.e. in the second decade of life (Sorenson et al., 1994). In contrast to the stimulated release of NO from the endothelium, the basal release of NO appears to be preserved in the peripheral vasculature of hypercholesterolemic patients as compared to normal individuals. It is important to note, that endothelial dysfunction of conduit and resistance vessels in the forearm is not associated with clinical symptoms in hypercholesterolemic patients. Since these patients often develop coronary artery disease, differences must exist in the forearm and coronary circulation. Studies in patients with hypercholesterolemia type IIa, moderately elevated serum cholesterol levels and angiografically smooth coronary arteries revealed a significant endothelial dysfunction of large coronary arteries and the coronary microcirculation. Although no coronary lesions can be detected by angiography in some of those patients intimal hyperplasia and structural alterations of the vascular wall are usually present in this setting, as assessed by intravascular ultrasound. Thus, the presence of coronary artery diseases underestimated by angiography. In hypercholesterolemic patients with angiographically smooth coronary arteries a
striking vasoconstriction in response to intracoronary infusion of acetylcholine can usually be observed. Since recent data suggest that the vasomotor response of acetylcholine is at least in part, mediated by the release of NO (Lefroy et al., 1993), the abnormal response to acetylcholine probably reflects the diminished availability of endothelial mediator. In keeping with experimental findings, endothelium-dependent relaxation of the coronary microcirculation is poorer in hypercholesterolemic than normocholesterolemic subjects (Drexler et al., 1991a; Zeiher et al., 1993) while endotheliumindependent vasodilators exert maximal increases in coronary blood flow in hypercholesterolemia. There is experimental evidence that under these circumstances, flow-dependent dilatation of the coronary microvasculature is abolished (Kuo et al., 1992). Since this mechanism of dilation of resistance vessels may be involved in the increased flow, i.e. during reactive hyperemia, the impaired endothelial function of the coronary microvasculature in humans may have important implications for subjects with hypercholesterolemia and endothelial dysfunction of the coronary microcirculation. Moreover, the endothelial modulation of factors such as the response to sympathetic stimulation or platelet products (thromboxane A22 and serotonin) is altered. With normal endothelial function, the release of NO may be associated with vasodilatation (counterbalancing the direct vasoconstrictor effects of serotonin and thromboxan). However, in the face of a defective endothelial mediated vasodilation, inappropriate coronary vasoconstriction may emerge promoting platelet activation and aggregation (Drexler et al., 1992; Shimokawa, 1998). In general, the degree of endothelial dysfunction of coronary microvasculature correlates with total serum cholesterol levels (Zeiher et al., 1993). However, if the analysis is confined to patients with hypercholesterolemia (rather than to patients with a wide range of serum cholesterol levels) total serum cholesterol levels do not correlate with endothelial function (Quyyumi, 1998). Notably, HDL and its efficiency of reverse cholesterol transport in subjects with normal and increased LDL levels (for review see Tall, 1990) may be an important determinant for endothelial function. Indeed, the ratio of LDL/ HDL rather than absolute values of LDL determined the degree of endothelial dysfunction in the coronary microcirculation, indicating that HDL exerts a protective effect on endothelial function in patients with hypercholesterolemia (Quyyumi, 1998). Moreover, plasma autoantibody levels against oxidized
Endothelial Dysfunction in Human Disease (a) Maximal ACH-induced flow (ml/min/100 ml)
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Figure 1 Relationship between maximal acetylcholineinduced blood flow and plasma levels of LDL (a) or autoantibodies against oxidized LDL (b). For (b), r= −0.53; n=32; P=0.002.
LDL are closely related to endothelium-dependent relaxations as tested by acetylcholine suggesting that oxidized LDL may more toxic to the endothelium than native LDL (Heitzer et al., 1996) (Fig. 1). The underlying mechanisms by which hypercholesterolemia impairs endothelial function in patients remains to be elucidated. Several possible mechanisms have been proposed including: a) reduced synthesis of endothelium-derived relaxing factor [EDRF]; b) altered membrane receptor coupling mechanisms affecting the release of EDRF and c) impaired diffusion or augmented destruction of EDRF in the vessel wall. Assuming that the intracellular availability of -arginine may become a rate-limiting factor for the production of nitric oxide
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and/or in hypercholesterolemia (i.e by inhibition of NO synthase by di-methyl--arginine [ADMA] or by alteration of the -arginine transporter), -arginine was given to patients with hypercholesterolemia and showed beneficial effects in several clinical studies (Drexler et al., 1991a; Craeger, 1992). Progressive impairment of endothelial function within the coronary circulation has been observed in patients (Zeiher et al., 1991) as assessed by acetylcholine and flow-dependent dilatation. In humans, angiographically smooth coronary arteries dilate in response to increased flow, reflecting flow-dependent dilation due to the release of NO. Flow-mediated dilation of coronary arteries is impaired only in the more advanced stages of coronary artery disease (Drexler et al., 1989b). The loss of flow-dependent dilation in atherosclerotic segments may favor vasoconstriction at sites where vasodilation could ordinarily occur upon an increase in flow. Such an impaired response to increased flow may in part explain the abnormal vasomotor responses to physiological stimuli in daily life such as exercise, mental stress or exposure to cold environment. Under normal conditions, flowmediated dilation represents the major mechanism by which the epicardial vessels respond to stimuli, e.g. exercise, that increase myocardial work and oxygen demand. EDRF-mediated, shear-induced dilatation opposes the myogenic vasoconstriction elicited by increases in pressure (Pohl et al., 1991; Jones et al., 1993). Since experimental data suggest that the endothelium plays an important role in maintaining vascular structure (Langille et al., 1986), the impaired basal release of atherosclerotic coronary arteries as demonstrated in vitro (Chester et al., 1990) may, therefore, have long-term implications concerning vascular remodelling of coronary arteries. Since NO interferes with the action and synthesis of endothelin, the lack of NO activity may favor the vascular expression of endothelin (Boulanger, 1990; Lu ¨ ¨ scher 1990; Lerman, 1992). Indeed, increased circulating and tissue endothelin immunoreactivity has been observed in patients with coronary artery disease (Lerman et al., 1991) and acute coronary syndromes (Wieczorek et al., 1994). Beyond epicardial coronary arteries coronary artery disease, advanced age and, in particular, hypercholesterolemia adversely affects endotheliummediated vasodilator function of the coronary resistance vessels. Although these functional alterations by itself are unlikely to reduce coronary blood flow to levels associated with myocardial ischemia, impaired endothelium-mediated function may potentiate known trigger mechanisms of myocardial ischemia. Physical activity or mental stress
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is associated with release of norepinephrine. In the presence of a functional endothelium, norepinephrine stimulates the release of EDRF via endothelial alpha2 receptors, thereby counterbalancing the vasoconstrictor effect of norepinephrine on the underlying vascular smooth muscle. In the face of endothelial dysfunction, the vessel is more sensitive to the constrictor effects of norepinephrine. Indeed, Vita et al. have shown that patients with evidence of coronary endothelial dysfunction as assessed by acetylcholine, intracoronary infusion of phenylephrine was associated with increased coronary vasoconstriction as compared to normal individuals (Vita et al., 1992). Similarly, exercise or exposure to cold activates the sympathetic nervous system and increases circulating levels of catecholamines. Sympathetic activation is also accompanied by complex reflex mechanisms including increases in heart rate and blood pressure thereby enhancing myocardial oxygen demand. In people with normal coronary arteries exercise and exposure to cold produce dilatation of epicardial coronary arteries (Gordon et al., 1989; Zeiher et al., 1989). In contrast, patients with arteriosclerotic coronary arteries demonstrate “paradoxical” epicardial coronary vasoconstriction in response to exercise, cold pressure test or mental stress (Gordon et al., 1989; Zeiher et al., 1989; Yeung et al., 1991). In such patients there is a close relationship between endothelial function and response of the same coronary segment to the cold pressure test. Coronary vasoconstriction is usually observed only in segments with acetylcholine-induced epicardial coronary vasoconstriction (indicating endothelial dysfunction) (Zeiher et al., 1989). There is evidence that exercise induces dynamic vasoconstriction at the site of arteriosclerotic lesions (Gage et al., 1986). Mental stress or cold exposure are other documented stimuli to induce myocardial ischemia in these patients. Conceivably, this inappropriate vasoconstriction is due to endothelial vasodilator dysfunction of atherosclerotic epicardial arteries and therefore, the dysfunctional endothelium may play an important role in triggering myocardial ischemia in patients with (intermediate) coronary stenoses. Even though the extent of coronary vasoconstriction elicited by sympathetic activation is usually less than 25–30%, such an increase in arterial tone may be sufficient to convert a nonflow-limiting into a critical stenosis with ensuing decreases in blood flow; i.e. in intermediate coronary lesions of 50–70%. Thus, in coronary lesions with stenoses between 50–70% (luminal diameter) an abnormal vasoconstrictor response due to the loss
of endothelium-mediated vasodilation may account for the mismatch between myocardial oxygen supply and demand, in other words, a link may exist between endothelial vasodilator dysfunction at the site of the stenosis and myocardial ischemia and explain episodes of angina associated with daily life activities.
Chronic heart failure In chronic heart failure, systemic compensatory mechanisms are activated to maintain arterial blood pressure in the face of reduced cardiac output. In particular, the sympathetic system and the renin– angiotensin system contribute to an increased systemic vasoconstriction (Zelis and Flaim, 1982). Yet, short-term alpha-adrenergic blockade or angiotensin converting enzyme-inhibition is not accompanied by increased blood flow to skeletal muscle during exercise indicating that factors other than norepinephrine or angiotensin II are involved in the impaired blood flow response during exercise (Drexler et al., 1989a; LeJemtel et al., 1986). Whereas neurohumoral factors have been studied extensively in the past, the impact of local factors regulating peripheral vasomotor tone and tissue perfusion in heart failure are poorly defined. Based on observations in animal models of heart failure, an impairement of endothelium-dependent vasodilatation may represent a local mechanism involved in the reduced metabolic vasodilation in patients with chronic heart failure. Several clinical studies have now studied endothelial function in patients with heart failure. By assessing the blood flow response to acetylcholine in the forearm or hindlimb, these studies have demonstrated an impaired increase in blood flow in patients with severe heart failure (Katz et al., 1992; Kubo et al., 1991; Drexler et al., 1991b). The impaired endotheliumdependent dilatation of forearm resistance vessels is impaired in heart failure suggests a decreased stimulated release of nitric oxide in response to acetylcholine. In contrast to the responses elicited by acetylcholine, the decrease in blood flow induced by N-monomethyl--arginine (-NMMA) was enhanced in patients with chronic heart failure (Drexler et al., 1991b); indicating that blockade of the basal release results in an exaggerated vasoconstrictor response in these patients. Similarly, following systemic administration of -NMMA, Habib et al. observed that the increase in systemic vascular resistance following -NMMA was greatest in those patients with heart failure who already had the highest basal systemic vascular resistance
Endothelial Dysfunction in Human Disease
(Habib et al., 1994). Moreover, Winlaw et al. measured plasma nitrate, the stable end-product of nitric oxide production, and reported increased levels in patients with heart failure (Winlaw et al., 1994). Taken in conjunction, these observations suggest, that the basal release of nitric oxide may be preserved or even enhanced in chronic heart failure. The vasomotor response to acetylcholine in patients with heart failure cannot be merely attributed to the release of EDRF. Acetylcholine-stimulated release of a cyclooxygenase-dependent vasoconstrictor substance has been reported previously in a femoral artery preparation from a canine model of heart failure (Kaiser et al., 1989). The reduced vasodilator response to acetylcholine in patients with chronic heart failure includes an abnormal production and release of a cyclooxygenase-dependent vasoconstrictor (Katz, 1993). Moreover, the vasodilatory response to nitroglycerin was reduced in patients with severe heart failure, suggesting a decreased responsiveness of vascular smooth muscle to cyclic GMP mediated vasodilation (Katz, 1993). The vasoconstrictor effects of acetylcholine on endothelium-mediated dilation of large conduit vessels (radial artery) in response to acetylcholine are modest and do not discriminate between normal subjects and patients with heart failure. However, flow-dependent dilation of peripheral conduit arteries during reactive hyperemia is reduced in these patients (Hayoz et al., 1993). Flow-dependent dilation is endothelium-dependent due to the release of EDRF (Hornig et al., 1996) and can be, in part, blocked by -NMMA in humans (Hornig et al., 1996). Endothelial dysfunction of conduit vessels may have functional consequences since it impairs the elastic properties of large conduit vessels and has a negative feedback on the pumping performance of the left ventricle (Ramsey and Jones, 1994). There is evidence that EDRF dynamically controls the distensibility of large arteries suggesting a physiological role of EDRF to reduce cardiac work relative to tissue perfusion by affecting arterial impedence (Ramsey et al., 1993). Large artery compliance is altered in patients with chronic heart failure (Arnold et al., 1991). Endothelial dysfunction may be involved in this abnormality. The functional importance of impaired endothelium-dependent relaxation of peripheral resistance vessels in tissue perfusion is still unsettled. If the stimulated release of nitric oxide during exercise is limited this may affect tissue perfusion during exercise (Loscalzo, 1994). It should be noted, however, that the physiological role of the release of EDRF during exercise in humans remains con-
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troversial; whereas some studies have reported decreased blood flow during reactive hyperemia or exercise following blockade of NO synthesis (Tagawa et al., 1994; Loscalzo and Vita, 1994; Gilligan et al., 1994b), others have not (Wilson and Kapoor, 1993). Yet, observations of Hirooka et al. suggest that -arginine improves the impaired acetylcholine induced and ischemic vasodilation in patients with heart failure, consistent with the notion that a defective endothelial function contributes to impaired vasodilator capacity in chronic heart failure (Hirooka et al., 1994). Circulating cytokines, and particularly, tumor necrosis factor (TNF) are increased in severe chronic heart failure (Levine et al., 1990). Experimental evidence suggests that TNF alpha is able to impair the stimulated release of EDRF (Aoki et al., 1989) and to impair the stability of the endothelial nitric oxide synthase mRNA (Yoshizumi et al., 1993). Katz et al. showed that plasma levels of tumor necrosis factor in patients with heart failure are related to the degree of endothelial dysfunction as tested by acetylcholine (Katz et al., 1994). Vice versa, TNF alpha may induce an inducible NO synthase in vascular smooth muscle or endothelium which, in turn may account for an increased basal release of nitric oxide (Lamas, 1991). Other potential mechanisms involved in the pathogenesis of endothelial dysfunction in heart failure include (a) chronically decreased blood flow (Miller and Vanhoutte, 1988) resulting in decreased expression of endothelial NO synthase and (b) increased tissue angiotensin-converting enzyme (ACE) activity resulting in increased breakdown of bradykinin. A bradykinin antagonist prevents the beneficial effect of ACEinhibition in normal volunteers, suggesting that ACE-inhibition improves endothelium-dependent dilation in response to increased flow by a bradykinin-dependent mechanism (Hornig et al., 1997). This observation after acute administration of ACE-inhibitors in humans however, does not preclude the possibility that endothelial dysfunction may be, in part, related to increased formation of oxygen-derived radicals. Increased ACE-activity can affect endothelium-dependent relaxation by enhancing the formation of angiotensin which, in turn may enhance local superoxide formation (Rajagopalan, 1996). In this respect hypercholesterolemia or angiotensin II has been shown to enhance the activity of NAD/NADPH oxidase, which is thought to represent the most potent source of superoxide anions (Rajagopalan, 1996). Moreover, it remains to be determined whether the beneficial role of bradykinin is persistent during longterm treatment, (c) reduced expression of endo-
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Table 1 Endothelial dysfunction in heart failure: potential mechanisms Increased levels of cytokines i.e. tumor necrosis factor impairing the synthesis of endothelial NO-synthase Increased angiotensin-converting enzyme activity enhancing the breakdown of bradykinin and/or increasing levels of oxygen radicals that inactivates EDRF/NO) Chronically reduced blood flow decreases of expression of endothelial NO-synthase Increased endothelial dependent vasoconstrictor agents i.e. cyclooxygenase-dependent factor counteract the vasodilating effects of nitric oxide Impaired endothel receptor-signal transduction pathways; i.e. dysfunctional muscarinic receptor Table 2 Potential therapeutic interventions Lipid-lowering drugs (hypercholesterolemia) Angiotensin-converting enzyme inhibition -arginine Radical scavengers (super oxide dismutase) Antioxidants (probucol, vitamin C and E, glutathione) Physical training Estrogens (postmenopausal women)
thelial NO synthase which is regulated by shear stress (Nishida, 1992). Thus, changes in flow should affect the expression of NO synthase. Indeed, reduced expression of endothelial NO synthase has been documented in experimental heart failure in dogs (Smith et al., 1996).
Therapeutic interventions Experimental studies have demonstrated complete restoration of endothelial function in monkeys fed an atherogenic diet followed by normal diet (Harrison et al., 1987). Improvement in endothelial function occurred before regression of the atherosclerotic lesions (Benzuly et al., 1994) suggesting that endothelial dysfunction is reversible if the primary insult, that is hypercholesterolemia, has been removed. Based on these experimental observations clinical studies have evaluated various interventions to restore endothelial dysfunction in humans (see Table 2). As outlined above, -arginine is the precursor of nitric oxide and may become a rate-limiting step in the synthesis of nitric oxide under pathophysiological conditions. While the underlying mechanism(s) remain to be elucidated arginine improves endothelial dysfunctions in some but not all cardiovascular diseases tested. Several long-term studies have applied lipid-lowering drugs for several months and evaluated endothelial function before and after this intervention
Percent change in diameter mediated by NO
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Figure 2 Change in radial artery diameter (%) during reactive hyperemia that can be blocked by -NMMA in normal individuals (Φ), patients with congestive heart failure (Ε), vitamin C acutely (∆) and after 4 weeks of oral therapy with vitamin C (Γ).
in patients with coronary artery disease and hypercholesterolemia (Leung et al., 1993; Egashira et al., 1994; Anderson et al. 1995; Treasure et al., 1995). Endothelium-dependent dilatation of coronary epicardial arteries improved significantly 6–12 months following a lipid-lowering regimen. In one of these studies, the beneficial effect in endothelial-dependent relaxations of the coronary circulation was observed predominantly following combined treatment with statins and the antioxidant probucol (Anderson et al., 1995). In one of these trials, the incidence of angina was reduced (Anderson et al., 1995). In this respect, Gould et al. demonstrated that short-term (3-month) cholesterol lowering decreased the size and severity of perfusion abnormalities as assessed by positron emission tomography after dipyradamole in hypercholesterolemic patients with coronary artery disease (Gould et al., 1994). Although endothelial function was not tested in this particular study, improvement of endothelial function may have contributed to increased flow-mediated endotheliumdependent arteriolar dilatation in response to the direct effect of dipyridamole. This would be similar to the improvement of endothelium-dependent arteriolar dilation in the coronary microvasculature by -arginine. Moreover, Andrews et al. suggested that only 4 weeks treatment with statins improves myocardial ischemia as tested by 48-h holter monitoring of the ST segments (Andrews et al., 1997). Beyond reduction of LDL-cholesterol, statins may improve endothelial dysfunction by upregulating endothelial NO synthase (Laufs et al., 1998). Future pharmacological attempts to counteract endothelial dysfunction in atherosclerosis and
Endothelial Dysfunction in Human Disease
hypercholesterolaemia may include the interference with intra/extracellular pathways to increase the synthesis and activity of nitric oxide. Several experimental data strongly suggest that the inactivation of nitric oxide is enhanced in hypercholesterolemia and arteriosclerosis, ie. by oxidized LDL and/or superoxid radical formation of diseased vessels. Recent data suggest that endothelial dysfunction of epicardial coronary arteries and peripheral arteries can be improved by vitamin C (Solzbach et al., 1997; Levine et al., 1996; Hornig et al., 1998) (Fig. 2) or reduced glutathione (Kugiyama, 1998) Other means for maintaining the activity of NO include the use of antioxidants such as vitamin E. While experimental studies have yielded promising effects, observations in some clinical studies did not reveal beneficial effects of vitamin E in hypercholesterolemic patients (McDowell et al., 1994; Gilligan et al., 1994a). However, both investigations suffer from various limitations. In one small double-blind, placebo-controlled study, patients were on simvastain before entering the trial and, although not statistically significant, were not well-matched with regard to serum cholesterol; plasma cholesterol levels were borderline in the group receiving vitamine E (240 mg/dl). The treatment period was two month only and the dose of vitamin may have been too low. Similarly, the treatment period in the second (uncontrolled) study was only 1 month and, therefore, may have missed a beneficial effect weeks to months after achieving reduction of oxidative susceptibility of LDL (Gilligan et al., 1994a). Indeed, longterm therapy with probucol over 1 year combined with lovastatin was more effective than lovastain given alone in improving endothelial dysfunction of epicardial coronary arteries (Anderson et al., 1995). Moreover, 4-month treatment with vitamin E in hypercholesterolemic patients who smoked and who had increased levels of autoantibodies against oxidzed LDL at baseline, improved endothelial dysfunction markedly. In fact, the improvement of endothelial function with vitamin E was significantly related to the decrease in the levels of autoantibodies against oxidized LDL (Heitzer et al., 1997). Other interventions to improve endothelial dysfunction in various cardiovascular disorders include supplementation of estrogens in postmenopausal women, physical exercise, i.e. for patients with chronic heart failure and/or coronary artery disease, fish oil and the administration of ACE-inhibitors. ACE-inhibitors have been shown to improve endothelial function in patients with heart failure (Drexler et al., 1995; Hornig et al., 1997) and patients with coronary artery disease (Mancini
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et al., 1996). Acute administration of ACE-inhibition improves endothelial function by a bradykinin-dependent mechanism. However, the beneficial effect of long-term ACE-inhibition appears to be, in part, related to reduced inactivation of nitric oxide (unpublished observations). In this respect there is some evidence that ACE-inhibition enhances other endothelial functions such as enhancing the release of tissue plasminogen activator and decreasing plasminogen activator inhibitor I (Vaughan et al., 1997; Tomiyama et al., 1998). Numerous clinical trials are now underway to evaluate interventions to improve endothelial dysfunction in patients. Beyond the demonstration of improved endothelial function, it will be crucial to achieve clinical benefit, i.e. increase tissue perfusion associated with reduction of clinical symptoms such as angina in patients with coronary artery disease or ameliorate muscular fatigue in patients with heart failure. Finally, the preventive effect of preserved endothelial function with respect to the development of atherosclerosis or the deterioration of systemic hypertension needs to be addressed. In most clinical studies, the assessment of endothelial function was achieved by a rather invasive and thorough approach. Whether or not the evaluation of endothelial function in the forearm (Celermajer et al., 1992) represents a useful surrogate to predict the development of atherosclerosis in humans (i.e. for the clinically more important coronary and cerebral circulation) is an open but a relevant question, given the limitations to evaluate the latter circulatory beds.
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of antioxidant vitamins on low density lipoprotein oxidation and impaired endothelium dependent vasodilation in patients with hypercholesterolemia. J Am Coll Cardiol 24: 1611–1617. G DM, P JA, K CM, W MA, C PR, Q AA, 1994b. The contribution of endothelium-derived nitric oxide to exercise-induced vasodilation. Circulation 90: 2853–2858. G JB, G P, N EG, F RD, Z J, M GH, A RW, S AP, 1989. Atherosclerosis influences the vasomotor response of epicardial coronary arteries to exercise. J Clin Invest 83: 1946–1952 G KL, M JP, G DI, H MJ, E RP, L R, D SJ, 1994. Short-term cholesterol lowering decreases size and severity of perfusion abnormalities by positron emission tomography after dipyridamole in patients with coronary artery disease. A potential noninvasive marker of healing coronary endothelium. Circulation 89: 1530–1538. H F, D D, C D, O CM, C JG, 1994. Enhanced basal nitric oxide production in heart failure: another failed counter-regulatory vasodilator mechanism? Lancet 344: 371–373. H D, A ML, F PC, H DD, 1987. Restoration of endothelium-dependent relaxation by dietary treatment of atherosclerosis. J Clin Invest 80: 1808–1811. H D, D H, M¨ T, H B, Z AM, J H, B HR, Z R, 1993. Flow-mediated arterial dilation is abnormal in congestive heart failure. Circulation 87: VII–92–6. H T, Y¨ -H S, L J, K S, M¨ T, J J, O M, D H, 1996. Cigarette smoking potentiates endothelial dysfunction of forearm resistance vessels in patients with hypercholesterolemia Circulation 93: 1346–1353. H Y-H S, W E, M¨ T, D H, 1997. Effect of vitamin E on endothelial function and plasma autoantibodies in patients with increased oxidative stress. Circulation (Suppl): I–417. H Y, I T, T T, S M, E T, A S-I, T A, 1994. Effects of -arginine on impaired acetylcholine induced and ischemic vasodilation of the forearm in patients with heart failure. Circulation 90: 658–668. H B, M V, D H, 1996. Physical training improves endothelial function in patients with chronic heart failure. Circulation 93: 210–214. H B, K C, D H, 1997. Role of bradykinin in mediating vascular effects of angiotensin-converting enzyme inhibitors in humans. Circulation 95: 1115– 1118. H B, A N, K C, D H, 1998. Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure. Circulation 97: 363–368. J CJH, K L, D MJ, C WM, 1993. Myogenic and flow-dependent control mechanisms in the coronary microcirculation. Basic Res Cardiol 88: 2–10. K SD, B L, S C, S JA, J G, G M, S S, N SD, F R, LJ TH, 1992. Impaired endothelium-mediated vasodilation in the peripheral vasculature of patients with congestive heart failure. J Am Coll Cardiol 19: 918–925.
Endothelial Dysfunction in Human Disease K S, 1993. Impaired acetylcholine-mediated vasodilation in patients with congestive heart failure, role of endothelium derived vasodilating and vasoconstricting factors, Circulation 88: 55–61. K SD, R R, B JW, S M, D L, B R, LJ TH, 1994. Pathophysiological correlates of increased serum tumor necrosis factor in patients with congestive heart failure. Relation to nitric oxide-dependent vasodilation in the forearm circulation. Circulation 90: 12–16. K L, S RC, O NB, 1989. Heart failure depresses endothelium-dependent responses in canine femoral artery. Am J Physiol 256: H962–H967. K SH, R TS, B AJ, W RE, H SM, 1991. Endothelium-dependent vasodilation is attenuated in patients with heart failure. Circulation 84: 1589–1596. K K, 1998. Intracoronary infusion of reduced glutathione improves endothelial vasomotor response to acetylcholine in human coronary circulation, Circulation 97: 2299–2301. K L, D MJ, C MS, C WM, 1992. Pathophysiological consequences of atherosclerosis extend into the coronary microcirculation. Restoration of endothelium-dependent responses by -arginine. Circ Res 70: 465–476. L BL, O’D F, 1986. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science 231: 405–407. L S, 1991. Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha. Am J Physiol C634–C64160. L U, F VL, P J, L JK, 1998. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97: 1129– 1135. L DC, C T, U NG, D GJ, M A, 1993. Effect of inhibition of nitric oxide synthesis on epicardial coronary artery caliber and coronary blood flow in humans. Circulation 88: 43–54. LJ TH, M CS, L D, C BJ, 1986. Failure to augment maximal limb blood flow in response to one leg versus two-leg exercise in patients with severe heart failure. Circulation 74: 245–51. L A, E BS, H JW, H DM, S SM, B JC, 1991. Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. N Engl J Med 325: 997–1001. L A, 1992. Inhibition of endothelium-derived relaxing factor enhances endothelin-mediated vasoconstriction. Circulation 85: 1894–1898. L WH, L CP, W CK, 1993. Beneficial effect of cholesterol-lowering therapy on coronary endothelium-dependent relaxation in hypercholesterolaemic patients. Lancet 341: 1496–1500. L B, K J, M L, F HM, P M, 1990. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 323: 236–241. L GN, F B, K SN, G MD, K JF, V JA, 1996 Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation 93: 1107–1113. L J, V JA, 1994. Ischemia, hyperemia, exercise, and nitric oxide. Complex physiology and complex molecular adaptations. Circulation 90: 2556–2559.
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L¨ TF, 1990. Interaction between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins. Circ Res 66: 1088–1094. M, JGB, H GC, M C et al., 1996. Angiotensin-converting enzyme inhibition with quinapril improves endothelial vasomotor dysfunction in patients with coronary arterty disease. Circulation 94: 258–265. M RS, 1994. Southwestern internal medicine conference: endothelial dysfunction and vascular disease. Am J Med Sci 307:378–389. MD IFW, B GM, ME J, Y IS, N DP, MV GE, B I, T ER, J GD, 1994. The effect of probucol and vitamin E treatment on the oxidation of low-density lipoprotein and forearm vascular responses in humans. Eur J Clin Invest 24: 759–765. M VM, V PM, 1988. Enhanced release of endothelium-derived factor(s) by chronic increases in blood flow. Am J Physiol 255: H446–H451. N K, 1992. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase, J Clin Invest 90: 2092–2096. P U, H K, H A, B E, 1991. EDRFmediated shear-induced dilation opposes myogenic vasoconstriction in small rabbit arteries. Am J Physiol 261: H2016–H2023. Q A., 1998. Effect of atherosclerosis on endothelium-dependent inhibition of platelet activation in humans, Circulation 98: 17–24. R S, 1996. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. J Clin Invest 97: 1916–1923. R MW, J CJH, 1994. Large arteries are more than passive conduits. Br Heart J 72: 3–4. R MW, J CJH, S W, L MJ, H AH, 1993. Endothelium-derived relaxing factor dynamically controls large artery distensibility in normal subjects (abstract). Eur Heart J 14: 274. S H, 1998. Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and atherosclerosis. Circ Res 64: 900–914. S CJ, S D, H C, R BS, Z X, Z G, X XB, K Y, P K, S WC, H TH, 1996 Reduced gene expression of vascular endothelial NO synthase and cyclooxygenase-1 in heart failure. Circ Res 78: 58–64. S U, H B, J M, J H, 1997. Vitamin C improves endothelial dysfunction of epicardial coronary arteries in hypertensive patients. Circulation 96: 1513–1519. S KE, C DS, G D, H G, B DJ, D JE, 1994. Impairment of endothelium-dependent dilation is an early event in children n with familial hypercholesterolemia and is related to the lipoprotein (a) level. J Clin Invest 93: 50–55. T T, I T, E T, S M, H Y, T A, 1994. Role of nitric oxide in reactive hyperemia in human forearm vessels. Circulation 90: 2285–2290. T AR, 1990. Plasma high density lipoproteins. Meta-
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bolism and relationship to atherogenesis. J Clin Invest 86:379–384. T H, K Y, M H, K T, Y H, K T, D N, 1998. Relationship between endothelial function and fibrinoysis in early hypertension. Hypertension 31 (part 2): 321–327. T CB, K JL, W WS, et al., 1995. Beneficial effects of cholesterol lowering therapy on the coronary endotheium in patients with coronary artery disease. N Engl J Med 332: 481–487. V DE, R JL, R PM, A JMO, M FJ, P MA, 1997. Effects of ramipril on plasma fibrinolytic balance in patients with acute anterior infarction. The HEART investigators. Circulation 96: 442–447. V JA, T CB, Y AC, V VI, F GM, F D, G P, S AP, 1992. Patients with evidence of coronary endothelial dysfunction as assessed by acetylcholine infusion demonstrate marked increase in sensitivity to constrictor effects of catecholamines. Circulation 85:1390–1397. W JR, K S, 1993. Contribution of endothelium-derived relaxing factor to exercise-induced vasodilation in humans. J Appl Physiol 75: 2740– 2744. W I, H WG, W DJ, L CA, F KAA, 1994. Raised plasma endothelin in unstable angina and non-Q wave myocardial infarction: relation to cardiovascular outcome. Br Heart J 72: 436–441.
W DS, S GA, K AM, S CG, S PM, M PS, 1994. Increased nitric oxide production in heart failure. Lancet 344: 373–374. Y AC, V VI, K DS, V JA, R TJ, G P, S AP, 1991. The effect of atherosclerosis on the vasomotor response of coronary arteries to mental stress. N Engl J Med 325: 1551–1556. Y M, P MA, B JC, L M-E, 1993. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res 73: 205–209. Z AM, D H, W¨ H, S B, J H, 1989. Coronaryy vasomotion in response to sympathetic stimulation in humans: importance of the functional integrity of the endothelium. J Am Coll Cardiol 14: 1181–1190. Z AM, D H, W¨ H, J H, 1991. Modulation of coronary vasomotor tone in humans. Progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation 83: 391– 401. Z AM, D H, S B, J H, 1993. Endothelium-mediated coronary blood flow modulation in humans: effects of age, atherosclerosis, hypercholesterolemia, and hypertension. J Clin Invest 92: 652–662. Z R, F SF, 1982. Alterations in vasomotor tone in congestive heart failure. Prog Cardiovasc Dis 24: 437–459.
Endothelial Dysfunction: a Novel Therapeutic Target
Endothelial Dysfunction in Human Disease Helmut Drexler and Burkhard Hornig Medizinische Hochschule Hannover (MHH) Carl-Neubergstr.1, 30625 Hannover, Germany H. D B. H. Endothelial Dysfunction in Human Disease. Journal of Molecular and Cellular Cardiology (1999) 31, 51–60. The vascular endothelium plays a key role in the local regulation of vascular tone by the release of vasodilator substances (i.e. endothelium-derived relaxing factor (EDRF=nitric oxide, NO) and prostacyclin) and vasoconstrictor substances (i.e. thromboxane A2, free radicals, or endothelin). Using either agents like acetylcholine or changes in flow to stimulate the release of EDRF (NO), clinical studies have revealed the importance of EDRF in both basal and stimulated control of vascular tone in large epicardial coronary arteries and in the coronary microcirculation. The regulatory function of the endothelium is altered by cardiovascular risk factors or disorders such as hypercholesterolemia, chronic smoking, hypertension or chronic heart failure. Endothelial dysfunction appears to have detrimental functional consequences as well as adverse longterm effects, including vascular remodelling. Endothelial dysfunction is associated with impaired tissue perfusion particularly during stress and paradoxical vasoconstriction of large conduit vessels including the coronary arteries. These effects may cause or contribute to myocardial ischemia. Several mechanisms may be involved in the development of endothelial dysfunction, such as reduced synthesis and release of EDRF or enhanced inactivation of EDRF after its release from endothelial cells by radicals or oxidized low-density lipoprotein (LDL). Increased plasma levels of oxidized LDL have been noted in chronic smokers and are related to the extent endothelial dysfunction, raising the possibility that chronic smoking potentiates endothelial dysfunction by increasing circulating and tissue levels of oxidized LDL. In heart failure, cytokines and/or reduced flow (reflecting reduced shear stress) may be involved in the development of endothelial dysfunction and can be reversed by physical training. Other mechanisms include an activated renin-angiotensin system (i.e. postmyocardial infarction) with increased breakdown of bradykinin by enhanced angiotensin converting enzyme (ACE) activity. There is evidence that endogenous bradykinin is involved in coronary vasomotor control both in coronary conduit and resistance vessels. ACE inhibitors enhance endothelial function by a bradykinin-dependent mechanism and probably also by blunting the generation of superoxide anion. Endothelial dysfunction appears to be reversible by administering -arginine, the precursor of nitric oxide, lowering cholesterol levels, physical training, antioxidants such as vitamin C, or 1999 Academic Press ACE inhibition. K W: Nitric oxide; Hypercholesterolemia; Heart failure; Flow-dependent dilation.
Introduction The evaluation of endothelial function in humans has focused primarily on endothelial-dependent vasomotion of conduit and resistance vessels, related to the release of nitric oxide (NO) while only recently other important functions of the endothelium have been studied (i.e. adhesive properties of the endothelium with respect to the interaction with leucocytes and platelets (for review see Bevilacqua, 1994) or factors involved in the homeostasis of thrombosis and fibrinolysis (for review see Meidell, 1994). In particular, clinical studies have evaluated vasomotor tone following changes in 0022–2828/99/010051+10 $30.00/0
flow or stimuli for the release of NO from the endothelium, such as acetylcholine, substance P or serotonin. The in vivo assessment of endothelial control of vascular tone in humans is hampered by several limitations and assumptions which need to be considered for the adequate interpretation of clinical studies (for review see Drexler, 1997) Vascular endothelium-dependent tone involves several factors such as nitric oxide, prostaglandins, endothelin or endothelium-derived hyperpolarizing factor. Up to now, the evaluation of endotheliumdependent regulation of vascular tone in patients with cardiovascular disorders focused on the role of NO. Numerous cardiovascular disorders have 1999 Academic Press
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been shown to be associated with altered endothelium-dependent relaxation such as atherosclerosis, diabetes mellitus, smoking, post-menopause state, hypertension, heart failure. Several reviews have discussed these disorders, including the impact of treatment (Drexler, 1997, Celermajer, 1997). This brief review focuses on coronary artery disease and heart failure.
Hypercholesterolemia and coronary artery disease In the clinical setting, hypercholesterolemia is characterized by long-lasting and moderate increases in serum cholesterol and serum cholesterol LDL rather than excessive plasma levels as applied in experimental studies. Nevertheless, clinical studies investigating the effects of hypercholesterolemia on endothelial function in the human circulation have, in general, confirmed the experimental observations that it is is associated with impaired endotheliumdependent vasodilation of coronary and peripheral conduit and resistance vessels (Chowienczyk et al., 1992; Creager et al., 1992; Casino et al., 1993), as tested by endothelium-dependent relaxations in response to acetylcholine or increased flow. In fact, endothelial dysfunction appears to emerge early in humans with hypercholesterolemia, i.e. in the second decade of life (Sorenson et al., 1994). In contrast to the stimulated release of NO from the endothelium, the basal release of NO appears to be preserved in the peripheral vasculature of hypercholesterolemic patients as compared to normal individuals. It is important to note, that endothelial dysfunction of conduit and resistance vessels in the forearm is not associated with clinical symptoms in hypercholesterolemic patients. Since these patients often develop coronary artery disease, differences must exist in the forearm and coronary circulation. Studies in patients with hypercholesterolemia type IIa, moderately elevated serum cholesterol levels and angiografically smooth coronary arteries revealed a significant endothelial dysfunction of large coronary arteries and the coronary microcirculation. Although no coronary lesions can be detected by angiography in some of those patients intimal hyperplasia and structural alterations of the vascular wall are usually present in this setting, as assessed by intravascular ultrasound. Thus, the presence of coronary artery diseases underestimated by angiography. In hypercholesterolemic patients with angiographically smooth coronary arteries a
striking vasoconstriction in response to intracoronary infusion of acetylcholine can usually be observed. Since recent data suggest that the vasomotor response of acetylcholine is at least in part, mediated by the release of NO (Lefroy et al., 1993), the abnormal response to acetylcholine probably reflects the diminished availability of endothelial mediator. In keeping with experimental findings, endothelium-dependent relaxation of the coronary microcirculation is poorer in hypercholesterolemic than normocholesterolemic subjects (Drexler et al., 1991a; Zeiher et al., 1993) while endotheliumindependent vasodilators exert maximal increases in coronary blood flow in hypercholesterolemia. There is experimental evidence that under these circumstances, flow-dependent dilatation of the coronary microvasculature is abolished (Kuo et al., 1992). Since this mechanism of dilation of resistance vessels may be involved in the increased flow, i.e. during reactive hyperemia, the impaired endothelial function of the coronary microvasculature in humans may have important implications for subjects with hypercholesterolemia and endothelial dysfunction of the coronary microcirculation. Moreover, the endothelial modulation of factors such as the response to sympathetic stimulation or platelet products (thromboxane A22 and serotonin) is altered. With normal endothelial function, the release of NO may be associated with vasodilatation (counterbalancing the direct vasoconstrictor effects of serotonin and thromboxan). However, in the face of a defective endothelial mediated vasodilation, inappropriate coronary vasoconstriction may emerge promoting platelet activation and aggregation (Drexler et al., 1992; Shimokawa, 1998). In general, the degree of endothelial dysfunction of coronary microvasculature correlates with total serum cholesterol levels (Zeiher et al., 1993). However, if the analysis is confined to patients with hypercholesterolemia (rather than to patients with a wide range of serum cholesterol levels) total serum cholesterol levels do not correlate with endothelial function (Quyyumi, 1998). Notably, HDL and its efficiency of reverse cholesterol transport in subjects with normal and increased LDL levels (for review see Tall, 1990) may be an important determinant for endothelial function. Indeed, the ratio of LDL/ HDL rather than absolute values of LDL determined the degree of endothelial dysfunction in the coronary microcirculation, indicating that HDL exerts a protective effect on endothelial function in patients with hypercholesterolemia (Quyyumi, 1998). Moreover, plasma autoantibody levels against oxidized
Endothelial Dysfunction in Human Disease (a) Maximal ACH-induced flow (ml/min/100 ml)
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Figure 1 Relationship between maximal acetylcholineinduced blood flow and plasma levels of LDL (a) or autoantibodies against oxidized LDL (b). For (b), r= −0.53; n=32; P=0.002.
LDL are closely related to endothelium-dependent relaxations as tested by acetylcholine suggesting that oxidized LDL may more toxic to the endothelium than native LDL (Heitzer et al., 1996) (Fig. 1). The underlying mechanisms by which hypercholesterolemia impairs endothelial function in patients remains to be elucidated. Several possible mechanisms have been proposed including: a) reduced synthesis of endothelium-derived relaxing factor [EDRF]; b) altered membrane receptor coupling mechanisms affecting the release of EDRF and c) impaired diffusion or augmented destruction of EDRF in the vessel wall. Assuming that the intracellular availability of -arginine may become a rate-limiting factor for the production of nitric oxide
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and/or in hypercholesterolemia (i.e by inhibition of NO synthase by di-methyl--arginine [ADMA] or by alteration of the -arginine transporter), -arginine was given to patients with hypercholesterolemia and showed beneficial effects in several clinical studies (Drexler et al., 1991a; Craeger, 1992). Progressive impairment of endothelial function within the coronary circulation has been observed in patients (Zeiher et al., 1991) as assessed by acetylcholine and flow-dependent dilatation. In humans, angiographically smooth coronary arteries dilate in response to increased flow, reflecting flow-dependent dilation due to the release of NO. Flow-mediated dilation of coronary arteries is impaired only in the more advanced stages of coronary artery disease (Drexler et al., 1989b). The loss of flow-dependent dilation in atherosclerotic segments may favor vasoconstriction at sites where vasodilation could ordinarily occur upon an increase in flow. Such an impaired response to increased flow may in part explain the abnormal vasomotor responses to physiological stimuli in daily life such as exercise, mental stress or exposure to cold environment. Under normal conditions, flowmediated dilation represents the major mechanism by which the epicardial vessels respond to stimuli, e.g. exercise, that increase myocardial work and oxygen demand. EDRF-mediated, shear-induced dilatation opposes the myogenic vasoconstriction elicited by increases in pressure (Pohl et al., 1991; Jones et al., 1993). Since experimental data suggest that the endothelium plays an important role in maintaining vascular structure (Langille et al., 1986), the impaired basal release of atherosclerotic coronary arteries as demonstrated in vitro (Chester et al., 1990) may, therefore, have long-term implications concerning vascular remodelling of coronary arteries. Since NO interferes with the action and synthesis of endothelin, the lack of NO activity may favor the vascular expression of endothelin (Boulanger, 1990; Lu ¨ ¨ scher 1990; Lerman, 1992). Indeed, increased circulating and tissue endothelin immunoreactivity has been observed in patients with coronary artery disease (Lerman et al., 1991) and acute coronary syndromes (Wieczorek et al., 1994). Beyond epicardial coronary arteries coronary artery disease, advanced age and, in particular, hypercholesterolemia adversely affects endotheliummediated vasodilator function of the coronary resistance vessels. Although these functional alterations by itself are unlikely to reduce coronary blood flow to levels associated with myocardial ischemia, impaired endothelium-mediated function may potentiate known trigger mechanisms of myocardial ischemia. Physical activity or mental stress
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is associated with release of norepinephrine. In the presence of a functional endothelium, norepinephrine stimulates the release of EDRF via endothelial alpha2 receptors, thereby counterbalancing the vasoconstrictor effect of norepinephrine on the underlying vascular smooth muscle. In the face of endothelial dysfunction, the vessel is more sensitive to the constrictor effects of norepinephrine. Indeed, Vita et al. have shown that patients with evidence of coronary endothelial dysfunction as assessed by acetylcholine, intracoronary infusion of phenylephrine was associated with increased coronary vasoconstriction as compared to normal individuals (Vita et al., 1992). Similarly, exercise or exposure to cold activates the sympathetic nervous system and increases circulating levels of catecholamines. Sympathetic activation is also accompanied by complex reflex mechanisms including increases in heart rate and blood pressure thereby enhancing myocardial oxygen demand. In people with normal coronary arteries exercise and exposure to cold produce dilatation of epicardial coronary arteries (Gordon et al., 1989; Zeiher et al., 1989). In contrast, patients with arteriosclerotic coronary arteries demonstrate “paradoxical” epicardial coronary vasoconstriction in response to exercise, cold pressure test or mental stress (Gordon et al., 1989; Zeiher et al., 1989; Yeung et al., 1991). In such patients there is a close relationship between endothelial function and response of the same coronary segment to the cold pressure test. Coronary vasoconstriction is usually observed only in segments with acetylcholine-induced epicardial coronary vasoconstriction (indicating endothelial dysfunction) (Zeiher et al., 1989). There is evidence that exercise induces dynamic vasoconstriction at the site of arteriosclerotic lesions (Gage et al., 1986). Mental stress or cold exposure are other documented stimuli to induce myocardial ischemia in these patients. Conceivably, this inappropriate vasoconstriction is due to endothelial vasodilator dysfunction of atherosclerotic epicardial arteries and therefore, the dysfunctional endothelium may play an important role in triggering myocardial ischemia in patients with (intermediate) coronary stenoses. Even though the extent of coronary vasoconstriction elicited by sympathetic activation is usually less than 25–30%, such an increase in arterial tone may be sufficient to convert a nonflow-limiting into a critical stenosis with ensuing decreases in blood flow; i.e. in intermediate coronary lesions of 50–70%. Thus, in coronary lesions with stenoses between 50–70% (luminal diameter) an abnormal vasoconstrictor response due to the loss
of endothelium-mediated vasodilation may account for the mismatch between myocardial oxygen supply and demand, in other words, a link may exist between endothelial vasodilator dysfunction at the site of the stenosis and myocardial ischemia and explain episodes of angina associated with daily life activities.
Chronic heart failure In chronic heart failure, systemic compensatory mechanisms are activated to maintain arterial blood pressure in the face of reduced cardiac output. In particular, the sympathetic system and the renin– angiotensin system contribute to an increased systemic vasoconstriction (Zelis and Flaim, 1982). Yet, short-term alpha-adrenergic blockade or angiotensin converting enzyme-inhibition is not accompanied by increased blood flow to skeletal muscle during exercise indicating that factors other than norepinephrine or angiotensin II are involved in the impaired blood flow response during exercise (Drexler et al., 1989a; LeJemtel et al., 1986). Whereas neurohumoral factors have been studied extensively in the past, the impact of local factors regulating peripheral vasomotor tone and tissue perfusion in heart failure are poorly defined. Based on observations in animal models of heart failure, an impairement of endothelium-dependent vasodilatation may represent a local mechanism involved in the reduced metabolic vasodilation in patients with chronic heart failure. Several clinical studies have now studied endothelial function in patients with heart failure. By assessing the blood flow response to acetylcholine in the forearm or hindlimb, these studies have demonstrated an impaired increase in blood flow in patients with severe heart failure (Katz et al., 1992; Kubo et al., 1991; Drexler et al., 1991b). The impaired endotheliumdependent dilatation of forearm resistance vessels is impaired in heart failure suggests a decreased stimulated release of nitric oxide in response to acetylcholine. In contrast to the responses elicited by acetylcholine, the decrease in blood flow induced by N-monomethyl--arginine (-NMMA) was enhanced in patients with chronic heart failure (Drexler et al., 1991b); indicating that blockade of the basal release results in an exaggerated vasoconstrictor response in these patients. Similarly, following systemic administration of -NMMA, Habib et al. observed that the increase in systemic vascular resistance following -NMMA was greatest in those patients with heart failure who already had the highest basal systemic vascular resistance
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(Habib et al., 1994). Moreover, Winlaw et al. measured plasma nitrate, the stable end-product of nitric oxide production, and reported increased levels in patients with heart failure (Winlaw et al., 1994). Taken in conjunction, these observations suggest, that the basal release of nitric oxide may be preserved or even enhanced in chronic heart failure. The vasomotor response to acetylcholine in patients with heart failure cannot be merely attributed to the release of EDRF. Acetylcholine-stimulated release of a cyclooxygenase-dependent vasoconstrictor substance has been reported previously in a femoral artery preparation from a canine model of heart failure (Kaiser et al., 1989). The reduced vasodilator response to acetylcholine in patients with chronic heart failure includes an abnormal production and release of a cyclooxygenase-dependent vasoconstrictor (Katz, 1993). Moreover, the vasodilatory response to nitroglycerin was reduced in patients with severe heart failure, suggesting a decreased responsiveness of vascular smooth muscle to cyclic GMP mediated vasodilation (Katz, 1993). The vasoconstrictor effects of acetylcholine on endothelium-mediated dilation of large conduit vessels (radial artery) in response to acetylcholine are modest and do not discriminate between normal subjects and patients with heart failure. However, flow-dependent dilation of peripheral conduit arteries during reactive hyperemia is reduced in these patients (Hayoz et al., 1993). Flow-dependent dilation is endothelium-dependent due to the release of EDRF (Hornig et al., 1996) and can be, in part, blocked by -NMMA in humans (Hornig et al., 1996). Endothelial dysfunction of conduit vessels may have functional consequences since it impairs the elastic properties of large conduit vessels and has a negative feedback on the pumping performance of the left ventricle (Ramsey and Jones, 1994). There is evidence that EDRF dynamically controls the distensibility of large arteries suggesting a physiological role of EDRF to reduce cardiac work relative to tissue perfusion by affecting arterial impedence (Ramsey et al., 1993). Large artery compliance is altered in patients with chronic heart failure (Arnold et al., 1991). Endothelial dysfunction may be involved in this abnormality. The functional importance of impaired endothelium-dependent relaxation of peripheral resistance vessels in tissue perfusion is still unsettled. If the stimulated release of nitric oxide during exercise is limited this may affect tissue perfusion during exercise (Loscalzo, 1994). It should be noted, however, that the physiological role of the release of EDRF during exercise in humans remains con-
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troversial; whereas some studies have reported decreased blood flow during reactive hyperemia or exercise following blockade of NO synthesis (Tagawa et al., 1994; Loscalzo and Vita, 1994; Gilligan et al., 1994b), others have not (Wilson and Kapoor, 1993). Yet, observations of Hirooka et al. suggest that -arginine improves the impaired acetylcholine induced and ischemic vasodilation in patients with heart failure, consistent with the notion that a defective endothelial function contributes to impaired vasodilator capacity in chronic heart failure (Hirooka et al., 1994). Circulating cytokines, and particularly, tumor necrosis factor (TNF) are increased in severe chronic heart failure (Levine et al., 1990). Experimental evidence suggests that TNF alpha is able to impair the stimulated release of EDRF (Aoki et al., 1989) and to impair the stability of the endothelial nitric oxide synthase mRNA (Yoshizumi et al., 1993). Katz et al. showed that plasma levels of tumor necrosis factor in patients with heart failure are related to the degree of endothelial dysfunction as tested by acetylcholine (Katz et al., 1994). Vice versa, TNF alpha may induce an inducible NO synthase in vascular smooth muscle or endothelium which, in turn may account for an increased basal release of nitric oxide (Lamas, 1991). Other potential mechanisms involved in the pathogenesis of endothelial dysfunction in heart failure include (a) chronically decreased blood flow (Miller and Vanhoutte, 1988) resulting in decreased expression of endothelial NO synthase and (b) increased tissue angiotensin-converting enzyme (ACE) activity resulting in increased breakdown of bradykinin. A bradykinin antagonist prevents the beneficial effect of ACEinhibition in normal volunteers, suggesting that ACE-inhibition improves endothelium-dependent dilation in response to increased flow by a bradykinin-dependent mechanism (Hornig et al., 1997). This observation after acute administration of ACE-inhibitors in humans however, does not preclude the possibility that endothelial dysfunction may be, in part, related to increased formation of oxygen-derived radicals. Increased ACE-activity can affect endothelium-dependent relaxation by enhancing the formation of angiotensin which, in turn may enhance local superoxide formation (Rajagopalan, 1996). In this respect hypercholesterolemia or angiotensin II has been shown to enhance the activity of NAD/NADPH oxidase, which is thought to represent the most potent source of superoxide anions (Rajagopalan, 1996). Moreover, it remains to be determined whether the beneficial role of bradykinin is persistent during longterm treatment, (c) reduced expression of endo-
H. Drexler and B. Hornig
Table 1 Endothelial dysfunction in heart failure: potential mechanisms Increased levels of cytokines i.e. tumor necrosis factor impairing the synthesis of endothelial NO-synthase Increased angiotensin-converting enzyme activity enhancing the breakdown of bradykinin and/or increasing levels of oxygen radicals that inactivates EDRF/NO) Chronically reduced blood flow decreases of expression of endothelial NO-synthase Increased endothelial dependent vasoconstrictor agents i.e. cyclooxygenase-dependent factor counteract the vasodilating effects of nitric oxide Impaired endothel receptor-signal transduction pathways; i.e. dysfunctional muscarinic receptor Table 2 Potential therapeutic interventions Lipid-lowering drugs (hypercholesterolemia) Angiotensin-converting enzyme inhibition -arginine Radical scavengers (super oxide dismutase) Antioxidants (probucol, vitamin C and E, glutathione) Physical training Estrogens (postmenopausal women)
thelial NO synthase which is regulated by shear stress (Nishida, 1992). Thus, changes in flow should affect the expression of NO synthase. Indeed, reduced expression of endothelial NO synthase has been documented in experimental heart failure in dogs (Smith et al., 1996).
Therapeutic interventions Experimental studies have demonstrated complete restoration of endothelial function in monkeys fed an atherogenic diet followed by normal diet (Harrison et al., 1987). Improvement in endothelial function occurred before regression of the atherosclerotic lesions (Benzuly et al., 1994) suggesting that endothelial dysfunction is reversible if the primary insult, that is hypercholesterolemia, has been removed. Based on these experimental observations clinical studies have evaluated various interventions to restore endothelial dysfunction in humans (see Table 2). As outlined above, -arginine is the precursor of nitric oxide and may become a rate-limiting step in the synthesis of nitric oxide under pathophysiological conditions. While the underlying mechanism(s) remain to be elucidated arginine improves endothelial dysfunctions in some but not all cardiovascular diseases tested. Several long-term studies have applied lipid-lowering drugs for several months and evaluated endothelial function before and after this intervention
Percent change in diameter mediated by NO
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16 14
P < 0.05 P < 0.05
12 10
P < 0.01
8 6 4 2 0
Normals
CHF
CHF
CHF
Figure 2 Change in radial artery diameter (%) during reactive hyperemia that can be blocked by -NMMA in normal individuals (Φ), patients with congestive heart failure (Ε), vitamin C acutely (∆) and after 4 weeks of oral therapy with vitamin C (Γ).
in patients with coronary artery disease and hypercholesterolemia (Leung et al., 1993; Egashira et al., 1994; Anderson et al. 1995; Treasure et al., 1995). Endothelium-dependent dilatation of coronary epicardial arteries improved significantly 6–12 months following a lipid-lowering regimen. In one of these studies, the beneficial effect in endothelial-dependent relaxations of the coronary circulation was observed predominantly following combined treatment with statins and the antioxidant probucol (Anderson et al., 1995). In one of these trials, the incidence of angina was reduced (Anderson et al., 1995). In this respect, Gould et al. demonstrated that short-term (3-month) cholesterol lowering decreased the size and severity of perfusion abnormalities as assessed by positron emission tomography after dipyradamole in hypercholesterolemic patients with coronary artery disease (Gould et al., 1994). Although endothelial function was not tested in this particular study, improvement of endothelial function may have contributed to increased flow-mediated endotheliumdependent arteriolar dilatation in response to the direct effect of dipyridamole. This would be similar to the improvement of endothelium-dependent arteriolar dilation in the coronary microvasculature by -arginine. Moreover, Andrews et al. suggested that only 4 weeks treatment with statins improves myocardial ischemia as tested by 48-h holter monitoring of the ST segments (Andrews et al., 1997). Beyond reduction of LDL-cholesterol, statins may improve endothelial dysfunction by upregulating endothelial NO synthase (Laufs et al., 1998). Future pharmacological attempts to counteract endothelial dysfunction in atherosclerosis and
Endothelial Dysfunction in Human Disease
hypercholesterolaemia may include the interference with intra/extracellular pathways to increase the synthesis and activity of nitric oxide. Several experimental data strongly suggest that the inactivation of nitric oxide is enhanced in hypercholesterolemia and arteriosclerosis, ie. by oxidized LDL and/or superoxid radical formation of diseased vessels. Recent data suggest that endothelial dysfunction of epicardial coronary arteries and peripheral arteries can be improved by vitamin C (Solzbach et al., 1997; Levine et al., 1996; Hornig et al., 1998) (Fig. 2) or reduced glutathione (Kugiyama, 1998) Other means for maintaining the activity of NO include the use of antioxidants such as vitamin E. While experimental studies have yielded promising effects, observations in some clinical studies did not reveal beneficial effects of vitamin E in hypercholesterolemic patients (McDowell et al., 1994; Gilligan et al., 1994a). However, both investigations suffer from various limitations. In one small double-blind, placebo-controlled study, patients were on simvastain before entering the trial and, although not statistically significant, were not well-matched with regard to serum cholesterol; plasma cholesterol levels were borderline in the group receiving vitamine E (240 mg/dl). The treatment period was two month only and the dose of vitamin may have been too low. Similarly, the treatment period in the second (uncontrolled) study was only 1 month and, therefore, may have missed a beneficial effect weeks to months after achieving reduction of oxidative susceptibility of LDL (Gilligan et al., 1994a). Indeed, longterm therapy with probucol over 1 year combined with lovastatin was more effective than lovastain given alone in improving endothelial dysfunction of epicardial coronary arteries (Anderson et al., 1995). Moreover, 4-month treatment with vitamin E in hypercholesterolemic patients who smoked and who had increased levels of autoantibodies against oxidzed LDL at baseline, improved endothelial dysfunction markedly. In fact, the improvement of endothelial function with vitamin E was significantly related to the decrease in the levels of autoantibodies against oxidized LDL (Heitzer et al., 1997). Other interventions to improve endothelial dysfunction in various cardiovascular disorders include supplementation of estrogens in postmenopausal women, physical exercise, i.e. for patients with chronic heart failure and/or coronary artery disease, fish oil and the administration of ACE-inhibitors. ACE-inhibitors have been shown to improve endothelial function in patients with heart failure (Drexler et al., 1995; Hornig et al., 1997) and patients with coronary artery disease (Mancini
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et al., 1996). Acute administration of ACE-inhibition improves endothelial function by a bradykinin-dependent mechanism. However, the beneficial effect of long-term ACE-inhibition appears to be, in part, related to reduced inactivation of nitric oxide (unpublished observations). In this respect there is some evidence that ACE-inhibition enhances other endothelial functions such as enhancing the release of tissue plasminogen activator and decreasing plasminogen activator inhibitor I (Vaughan et al., 1997; Tomiyama et al., 1998). Numerous clinical trials are now underway to evaluate interventions to improve endothelial dysfunction in patients. Beyond the demonstration of improved endothelial function, it will be crucial to achieve clinical benefit, i.e. increase tissue perfusion associated with reduction of clinical symptoms such as angina in patients with coronary artery disease or ameliorate muscular fatigue in patients with heart failure. Finally, the preventive effect of preserved endothelial function with respect to the development of atherosclerosis or the deterioration of systemic hypertension needs to be addressed. In most clinical studies, the assessment of endothelial function was achieved by a rather invasive and thorough approach. Whether or not the evaluation of endothelial function in the forearm (Celermajer et al., 1992) represents a useful surrogate to predict the development of atherosclerosis in humans (i.e. for the clinically more important coronary and cerebral circulation) is an open but a relevant question, given the limitations to evaluate the latter circulatory beds.
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